The natriuretic system, a key mechanism in the maintenance of vascular tone and cardiovascular homeostasis, also plays a key role in regulation of the skeleton (Chusho et al. 2001a; Matsukawa et al. 1999; Suda et al. 1999). The mammalian natriuretic system consists of three related natriuretic peptides (NPs), ANP, BNP and CNP (Levin et al. 1998) and three receptors mediating the biological activity of these peptides: GC-A and GC-B which are coupled to guanylate cyclases, producing cGMP as a secondary messenger (Matsuo 2001; Hirose et al. 2001), and NPR-C which acts as a clearance receptor and is not linked to guanylate cyclase. The GC-A receptor preferentially binds ANP and BNP, and the GC-B receptor has CNP for cognate ligand. The third receptor, NPR-C, binds all three NPs with similar affinity (Suga et al. 1992). CNP- and BNP-transgenic mice and NPR-C knockout mice have elongated bones and marked kyphosis whereas CNP-knockout mice exhibit dwarfism. Prior to the present invention, no specific endogenous ligand had been identified for NPR-C, and it is thought to act mainly as a clearance receptor (Levin 1993). However, other biological functions have been postulated for this receptor (Levin 1993).
It is generally recognized that ANP and BNP are functionally distinct from CNP. Secretion of the former represents chronic (ANP) and acute (BNP) adaptive responses to elevated blood pressure. These molecules directly act on kidney glomerular and tubular cells to increase salt and water excretion, thereby leading to volume depletion and lowering of blood pressure. On the other hand, injection of physiological doses of CNP triggers minimal diuresis and natriuresis. The cardiovascular effects of CNP are characterized as a reduction in cardiac filling pressure and output, secondary to a direct effect on the vasculature. A further distinction between ANP/BNP and CNP concerns their range of action. ANP and BNP are considered classical endocrine regulators; the fact that both CNP and its receptor are produced locally in many tissues has lead to the suggestion that CNP is primarily a paracrine/autocrine factor. This notion has been reinforced by recent studies showing that bone-derived CNP is an important regulator of skeletal development.
Osteocrin (Ostn) is a recently discovered novel bone secreted protein with prohormone like characteristics (Thomas et al. 2003). The sequence of the protein was found to consist of 133 amino acids in human (SEQ ID NO: 1) and 130 (SEQ ID NO: 2) amino acids in mouse. It is produced by cells of the osteoblast lineage. Prior to the present invention, a specific function for Ostn had not been established, Ostn having no strong homology with any known protein family evident from in silico sequence analysis. However, limited C-terminal homology was recently observed with members of the natriuretic peptide family.
The best conserved homology between Ostn and the natriuretic peptides includes the residues Phe7, Gly8 and Arg13 (numbering according to CNP) that have been demonstrated to be necessary for peptide binding to the NPR-C receptor (Koyama et al. 1994; He et al. 2001; Veale et al. 2000). However, the lack of the two cysteine residues present in all NPs, suggests Ostn does not form the cyclic ring structure that is essential for binding to the receptors signalling through cGMP, GC-A and GC-B (Misono et al. 1984; Hirata et al. 1985a; Hirata et al. 1985d; Hirata et al. 1985c; Hirata et al. 1985b). Interestingly, synthetic ring-deleted, linear analogues of the NPs such as des-Cys105 have been shown to be specific ligands of the NPR-C receptor (Veale et al. 2000; Koyama et al. 1994; Olins et al. 1988; Smyth & Keenan 1994; Maack et al. 1987).
Currently a significant body of literature exists demonstrating a role for the natriuretic system in regulation of the skeleton. In NPR-C knockout mice (Jaubert et al. 1999; Matsukawa et al. 1999) as well as in BNP- and CNP-overexpressing mice (Suda et al. 1998; Miyazawa et al. 2002) bone overgrowth presumably correlated with increased NP bioavailability was observed. Further, presence of the GC-A and GC-B receptors and production of cGMP in response to NPs has been well-established in both osteoblasts (Fletcher et al. 1986; Yanaka et al. 1998; Nashida et al. 1996; Hagiwara et al. 1996b; Inoue et al. 1996a; Inoue et al. 1996b; Fletcher et al. 1986; Suda et al. 1996) and chondrocytes (Suda et al. 2002; Yamashita et al. 2000; Fujishige et al. 1999; Hagiwara et al. 1996a; Hagiwara et al. 1994).
However, to date a role for NPR-C-specific antagonists has not been demonstrated within the skeleton. Two studies have investigated the action of specific NPR-C antagonists in ex vivo bone systems. In a foetal mouse tibial organ culture assay, treatment with CNP resulted in significant increases in bone length associated with increases in cGMP accumulation. When the bones were treated with the NPR-C antagonist C-ANF no effect was apparent (Yasoda et al. 1998). Similarly, in primary rat osteoblastic cultures, both ANP and CNP inhibited proliferation and stimulated osteoblast differentiation whereas C-ANF treatment had no effect on osteoblast differentiation (Hagiwara et al. 1996b).
There remains a need to identify specific NPR-C ligands capable of modulating local levels of NPs and promoting osteogenesis.
The identification of specific NPR-C ligands might advantageously have a more specific effect on bone metabolism avoiding cardiovascular side-effects.
The present invention seeks to meet these needs and other needs.
The present description refers to a number of documents, the content of which is herein incorporated by reference in their entirety.